WO2016127007A2 - Capteur à nanopore comprenant un passage fluidique - Google Patents

Capteur à nanopore comprenant un passage fluidique Download PDF

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WO2016127007A2
WO2016127007A2 PCT/US2016/016664 US2016016664W WO2016127007A2 WO 2016127007 A2 WO2016127007 A2 WO 2016127007A2 US 2016016664 W US2016016664 W US 2016016664W WO 2016127007 A2 WO2016127007 A2 WO 2016127007A2
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nanopore
fluidic
passage
reservoir
electrical potential
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PCT/US2016/016664
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WO2016127007A3 (fr
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Ping Xie
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President And Fellows Of Harvard College
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Priority to CN202010965386.5A priority Critical patent/CN112816679B/zh
Priority to EP23191347.6A priority patent/EP4293349A3/fr
Priority to CN201680019980.7A priority patent/CN107533045B/zh
Priority to JP2017540674A priority patent/JP6800862B2/ja
Priority to EP16706099.5A priority patent/EP3254103B1/fr
Publication of WO2016127007A2 publication Critical patent/WO2016127007A2/fr
Publication of WO2016127007A3 publication Critical patent/WO2016127007A3/fr

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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
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    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
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    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
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    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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    • G01N27/416—Systems
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    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
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    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/4473—Arrangements for investigating the separated zones, e.g. localising zones by electric means
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    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0896—Nanoscaled
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0421—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electrophoretic flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4146—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS involving nanosized elements, e.g. nanotubes, nanowires

Definitions

  • This invention relates generally to sensing systems that employ a nanopore sensor, and more particularly relates to techniques fo sensing species as the species translocate a nanopore sensor.
  • Bot solid-state nanopores and biological nanopores are
  • a common approach in nanopore-based sensing employs the measurement of ionic current flow through a nanopore that is provided in a highly resistive amphiphilic membrane between electrodes provided, on either side of the membrane.
  • a molecule such as a polymer analyte like DNA is caused to translocate the nanopore, the ionic current flow through the nanopore is modulated, by the different nucleotide bases of a DNA strand.
  • Measurement in changes in ionic current flow can be carried out in order to determine a sequence characteristic of the polymer strand.
  • Nanopore devices for detection of anaiytes other than polynucleotides have also been reported, for example in International Patent Application PCT/TJS2013/028414, published as WO2013 123379, for the detection of proteins. Whilst there has also been considerable effort in developing methods and systems using solid state nanopores in order to sequence DNA, there remain a host of challenges for commercial realization. In addition, various configurations of nanopores pose particular challenges. For example, in the use of an array of nanopores in which ionic current flow through each nanopore i the array can be measured, a measurement can be made between a common electrode and a plurality of electrodes provided on the respective opposite side of each nanopore. Here the plurality of electrodes needs to be electrically isolated from each other, limiting the level of
  • Biological nanopores in some respects are advantageous over solid state nanopores in that they provide a constant and reproducible physical aperture.
  • the amphophilic membranes in which they are provided are in general fragile and may be subject, to degradation, providing ionic leakage pathways through the membrane.
  • the speed of translocation of an analyte through a biological nanopore can be controlled by the use of an enzyme.
  • Enzyme-assisted translocation of polynucleotides is typically on the order of 30 bases/second. In order to increase the throughput rate of analyte, much higher translocation speeds are desirable, but it is found that in general, the measurement of the a sensing signal can be problematic.
  • nanopore sensing methods are in general directed to an arrangement in which there is recorded relativel local nanopore signals employing electronic sensors that are integrated with the nanopore.
  • These nanopore sensing methods include, e.g., measurement of capacitive coupling across a nanopore and tunnelling current measurements through a species translocating a nanopore. While providing interesting alternative sensing techniques, such capacitive coupling and tunnelling current measurement techniques have not yet improved upon the conventional ionic current detection technique for nanopore sensing, and ionic current detection, techniques remain challenged by signal amplitude and signal bandwidth, issues,
  • the nanopore senso includes a. nanopore disposed in a support structure.
  • a fluidie passage is disposed between a first fluidie reservoir and the nanopore to fluidically connect the first fluidie reservoir to the nanopore through the fluidie passage.
  • the fluidie passage has a passage length that is greater than the passage width.
  • a second fluidie reservoir is fluidically connected to the nanopore, with the nanopore providing fluidie communication between the fluidie passage the second reservoir.
  • Electrodes are connected to impose a electrical potential difference across the nanopore.
  • At least one electrical transduction element is disposed in the nanopore sensor with a connection to measure the electrical potential that is local to the fluidie passage.
  • This nanopore sensor configuration enables local electrical potential sensing by a transduction element to provide high sensitivity, high bandwidth, and localized large signal proportional to the ionic current.
  • nanopore sensing applications such as DMA sequencing can be accomplished with the nanopore sensor at a very high integration density and throughput of analyte.
  • FIG. 1A is a schematic circuit diagram of a first example nanopore sensor configuration for measuring local electrical potential
  • FIG. IB is a circuit diagram- of an example transistor
  • FIG. 1C is a schematic circuit diagram of a second example nanopore sensor configuration for measuring a local electrical potential
  • [00121 Fig. ID is circuit diagram of an example transistor
  • IE is a circuit diagram of an example transistor
  • Fig. IF is a schematic plan view of a single electron transistor implementation of a nanopore sensor configuratio fo measuring local electrical potential
  • FIG. iG is a schematic plan view of a quantum point contact implementation of a nanopore sensor configuration for measuring local electrical potential
  • FIG. 1H is a schematic side view of a lipid hilayer including fluorescent dye arranged for implementation of a protein nanopore sensor configuration for measuring local electrical potential;
  • FIG. 2A is a schematic diagram and corresponding circuit elements for a nanopore sensor configuration for measuring local electrical potential
  • FIG. 2B is a circuit diagram for the nanopore sensor transistor implementation of Fig. IB;
  • FIG. 3A is a schematic side view of the geometric features of a nanopore sensor configuration for measuring local electrical potential as- defined for quantitative analysis of the sensor;
  • [OO 0J Figs. 8A-3B are plots of the electrical potential in a nanopore of a nanopore sensor for measuring local electrical potential, here plotted as a function of distance from the nanopore into the cis reservoir, for a
  • the cis and trans reservoirs include fluidic solutions of equal ionic concentration and for a configuration in which the cis and irons reservoirs include fluidic solutions of unequal ionic concentration, respectively;
  • Figs. 3D-3E are plots of the electrical field in a nanopore of a nanopore sensor for measuring local electrical potential correspondin to the plots of electrical potential of Figs, 3A-3B, respectively;
  • Fig. 4A is a plot of the change in potential in a nanopore for a 50 nm- thick nanopore membrane and a configuration of a 1 V transmembrane voltage (TMV) for electrophoretic species translocation as a dsDNA molecule translocates through the nanopore, as a function of the CaJCir
  • TMV transmembrane voltage
  • Fig. 4B is a plot of the change in potential in the tra reservoir for a 10 nm-diameter nanopore at 1 V TMV for the conditions of the plot of Fig. 4A;
  • ⁇ g- 4C is a plot of noise sources and signal as a function, of recording bandwidth for a nanopore sensor configured for local electrical potential measurement;
  • Fig, 4D is a plot of the bandwidth of a nanopore sensor configured for local electrical potential measurement as a function of cis chamber solution concentration for a range of reservoir solution concentration ratios
  • Fig, 4E is a plot of signal decal length from the nanopore site in a nanopore configured for local electrical potential measurement as a function of eis and trans reservoir solution concentration ratio
  • [00&7J Fig. 5 is a schematic view of a nanopore sensor including a fiuidic passage connected betwee a first reservoir, here the trans reservoir, and nanopore in a support structure;
  • FIG. 6 is a circuit model of the of nanopore sensor of Fig, 5;
  • FIG. 7 is a plot of the ratio of measured transconductanee signal to maximum achievable transconductanee signal as a function of the ratio between the fSuidic passage resistance and the nanopore resistance;
  • FIG. 8 is a schematic side view of the nanopore sensor of Fig. 5 with the definition of geometric parameters;
  • Fig. 9 is a plot of the ratio of resistance of the fiukiie passage to resistance of the nanopore in the sensor of Fig. 5 as a function of ratio of fluidi passage diameter to fluidic passage length, for selected sensor dimensions;
  • Big. 10 is a plot of the of the ratio of measured transconductanee signal to maximum achievable transconductanee signal as a function of the ratio fluidic passage diameter to fluidic passage length, for selected sensor dimensions;
  • Pig- 11 is a schematic side view of a first fluidic passage configuration
  • FIG. 12 is a schematic side view of a fluidic passage disposed on a support structure for a nanopore
  • [00351 B g. 18 is a schematic side view o a anodized aluminum oxide fluidic passage configuration
  • FIG. 14 is a schematic side view of a lateral fluidic passage configuration
  • FIG. 16A-I6E are schematic side views of fliiidic passage configurations arranged with elements for makin a local electrical potential measurement.
  • Fig. 17 is a schematic view of a nanopore sensor configured for local electrical potential measurement with a nanowire FET disposed on a membrane;
  • Fig. 18 is a perspective view of one example implementation of the nanopore sensor configuration of Fig, 17;
  • Figs. 19A-19B are a schematic view of a nanopore sensor configured for local electrical potential measurement with a nanowire FET disposed on a graphene membrane, and a plan view of an example
  • Figs. 20A-2OB are a schematic view of a nanopore sensor configured for local electrical potential measurement with a graphene layer disposed on a nanowire FET, and a plan view of an example implementation of this nanopore sensor, respectively;
  • Figs, 21A-21B are a schematic view of a nanopore sensor configured for local electrical potential measurement with a graphene membrane, and a plan view of an example implementation of this nanopore sensor, respectively;
  • Figs. 22A-22D are schematic plan views of example locations of a nanopore with respect to a nanowire in a nanopore sensor configured for local electrical potential measurement;
  • Fig. 23 is a plot of the sensitivity of a nanowire in a nanopore sensor configured for local electrical potential measurement before and after formation of a nanopore at the nanowire location;
  • Fig. 24A is a plot of ⁇ measured ionic current through a nanppore and ii)measiired nanowire FET conductance, respectively, as DNA translocates through a nanopore in a nanopore sensor configured for local electrical potential measurement, for a TMV of 2 V and 100:1 cisi trans reservoir solutio concentration ratio, with a local potential measurement made in the trans reservoir;
  • Fig. 24B is a plot of i) measured ionic current through a nanopore and u ⁇ measured nanowire FET conductance, respectively, as DNA translocates through a nanopore in a nanopore sensor configured for local electrical potential measurement, for a TMV of 2.4 V and 100:1 eisftrans reservoir solution concentration ratio, with a local potential measurement made in the trans reservoir;
  • Fig. 24G is a plot of i)measured ionic current through a nanopore and ii)measured nanowire FET conductance, respectively, as DNA translocates through a nanopore in a nanopore sensor configured for local electrical potential measurement, for a TMV of 0.6 V and 1:1 eisftrans reservoir solution concentration ratio, with a local potential measurement made in the trans reservoir; and
  • Fig- 25 is a plot of i) total ionic current measured through three nanopores sharing reservoirs, w)measured nanowire FET conductance through the first of the nanopores, w)measured nanowire FET conductance through the second of the nanopores, and r * r * )meas red nanowire FET conductance through the third of the nanopores, respectively .
  • DNA translocates through the nanopores in the three sensors in a nanopore sensor configured for local electrical potential measurement.
  • FIGS. 1A-1E are schematic views of nanopore sensor
  • a nanopore sensor 3 including a support, structure 14, suc as a membrane. In which is disposed a nanopore 12.
  • the nanopore 12 i configured in the support structure between two fluidic reservoirs shown here
  • nanopore 1.2 is the only path of fluidic communication between the cis and trans reservoirs.
  • One reservoir is connected to an inlet to the nanopore while the other reservoir is connected to an outlet from the nanopore.
  • one or more objects of a species such as molecules, are provided in a fluidic solution in one of the reservoirs for translocation through the nanopore to the other of the two reservoirs.
  • the species objects to be translocated through the nanopore can include objects selected from, fo example, DMA, DNA fragments, BNA, UNA fragments. PNA, nucleotides, nucleosides, oligonucleotides, proteins, polypeptides, amino acids and polymers.
  • the species objects can include a tag that is released from a tagged nucleotide.
  • nucleotides along a nucleic acid molecule can be polymerized to generate a nucleic acid strand that is complementary to at least a portion of the nucleic acid molecule, whereby, during polymerization, a tag is released from an individual nucleotide of the nucleotides, and whereb the released tag translocates the nanopore, as described in WO 2013/101 ' 793, hereby
  • the nanopore may he provided as an aperture, gap, channel, groove, pore or other hole in the suppor structure and is provided with an extent, such as a diameter, for corresponding geometry, that is suitable for sensin species objects of interest.
  • a nanopore of less than about 100 nm can be preferred, and a nanopore of less than 10 nm, 5 nm, or 2 nm can be more preferred.
  • a nanopore of 1 nm ca be suitable and even preferred for some molecular sensing applications,
  • the reservoirs or other components of the nanopore sensor may be configured to provide a driving force for moving objects of a species, such as molecules, toward the nanopore or through the nanopore from one of the reservoirs to the other of the reservoirs.
  • electrodes 3, 15 can be provided in a circuit with voltage and current elements 16, 18 to produce an electrophoretie force between the reservoirs for electrophoreticaiiy driving the species in the solution., towards the nanopore or through the nanopore from one reservoir to the other reservoir.
  • the fluidic solutions of the reservoirs can be provided as electrically conductive ionic solutions having pH and other characteristics that are amenable to the species i the solution.
  • an electrical circuit can he connected with the reservoir solutions in series through the nanopore, with electrodes 13, 15 as shown in the figures, providing an electrical voltage bias between the solutions, across the nanopore.
  • Translocation and control of the rate of translocation of species though the nanopore can be carried out with alternative techniques, such as an enzyme molecular motor,
  • a pressure gradient across the pore can be used to bring molecules towards the nanopore and/or through the nanopore.
  • This pressure gradient can be produced by using a physical pressure, or a chemical pressure such as an osmotic pressure.
  • An osmotic pressure can be produced from a concentration difference across the cis and trans chambers.
  • the osmotic pressure can be produced by having a concentration gradient of an osmotically active agent, such as a salt, polyethelene glycol (PEG), or glycerol.
  • a transduction element 7 that senses the electrical potential local to the site of the element and that develops a characteristic that is indicative of that local electrical potential.
  • An electrical connection such as device or region of a device and/or circuit, a wire, or combination of circuit elements, that senses the electrical potential local to the site of the device and/or circuit can be provided as a transduction element 7, to develop a signal indicative of local electrical potential.
  • the location of the electrical potential sensing can be in a reservoir, on a surface of the support, structure, or other location within the nanopore sensor as described in detail below.
  • a circuit 20 that includes, e.g., a transistor device 22, . having a source, 5, a drain, I ) , and a channel region 24.
  • the channel region 24 is in this example physically disposed at a location in the nanopore sensor environment to make a local electrical potential measurement. This physical location of the channel region 24 of the transistor can be at any convenient and. suitable site for accessing local electrical potential.
  • an electrical potential sensing circuit is configured local to the . rans reservoir to provide a transistor or other device that measures the electrical potential local to the trans reservoir at the trans reserooir-sida.of the nanopore 12.
  • an electrical transduction element 7 such as an electrical potential sensing device or circuit, can be configured at the cis reservoir side of the nanopore.
  • a circuit 20 including a transistor 24 or other device for measuring electrical potential local to the cis reservoir at the cis reservoir side of the nanopore 12.
  • Fig. IE there can be included two or more transduction elements, with circuits 20a, 20b, etc., connected to transduction elements such as transistors 22a, 22 that sense the electrical potential at two or more locations in the nanopore sensor system, such as each side of the nanopore support ' structure.
  • transduction elements such as transistors 22a, 22 that sense the electrical potential at two or more locations in the nanopore sensor system, such as each side of the nanopore support ' structure.
  • the electrical potential at the two sides of the nanopore membrane 14 can thereby be measured with this arrangement.
  • This is an example configuration in which is enabled a measurement of the difference in local potential between two sites in the nanopore sensor. It is therefore intended, that the term
  • measured local electrical potential refers to the potential at a single site in the nanopore sensor, refers to a difference or sum in local electrical potential betwee two or more sites, and refers to a local potential at two or more sites n the nanopore senso configuration.
  • the local electrical potential measurement can be made by any suitable device and/or circuit or other transduction element, including
  • a transduction element on the support structure 14 that is configured as a single electron transistor (SET) circuit 27.
  • the source, S f and drain, D, regions of the SET are disposed on the support structure, providing tunneling barriers to the SET 27.
  • the electrical conductance through the SET 27 depends on the energy level of the SET with respect to the Fermi level of the source, S, and drain, D.
  • the electrical potential, and corresponding energy level, of the SET changes as species objects translocate through the nanopore, changing the conductance of the SET circuit.
  • a quantum point contact (QPC) system 29 for making a local electrical potential measurement.
  • QPC quantum point contact
  • an electrically conductive region 31 is provided that forms source. S, and drain, D, regions that are connected via a very thin conducting channel region at the site of the nanopore 12.
  • the channel region is sufficiently thin that the electronic carrier particle energy states that are perpendicular to the channel region are quantized.
  • the local potential around the QPC thus the Fermi level inside the thin conduction channel region changes, resulting in a change in the number of quantized states below the Fermi level, and a corresponding change in QPC conductance.
  • a nanowire FET can also be configured at the site of the
  • nanopore The nanowire can be formed of any suitable electrically conducting or semiconducting material, including fuilerene structures and semiconducting wires.
  • nanowire refers to an electrical conduction channel that is characterized by a width that is compatible with the signal decay length measured from the nanopore site. The channel width is preferably on the same order of magnitude as the decay length and can be larger.
  • the nanowire can be made from any semiconductor material that is stable in the selected reservoir solution.
  • the nanopore sensor is not limited to solid state nanopore configurations with solid state voltage sensing devices.
  • Biological nanopores and potential sensing arrangements can also be employed, e.g., with a protein nanopore or other suitable configuration.
  • an amphiphilic layer 31 in which is disposed a protein nanopore 33.
  • a voltage-sensitive dye e.g.. a fluorescent direct dye 37, can be provided in the lipid Mayer as an electrical transduction element.
  • Optical detection or sensing of the dye fluorescence and changes to that fluorescence provide sensing of the electrical potential at the nanopore.
  • Optical microscopy or other conventional arrangement can be employed for making this potential measurement as an optical output signal from the nanopore sensor.
  • This amphiphilic layer nanopore sensor is an example of a biological nanopore sensor that is based on sensing of the local potential at a site in the nanopore system.
  • the method of local potential measurement fo nanopore translocation detection is not limited to a particular solid state or biological configuration and can be applied to an suitable nanopore configuration.
  • the support structure can be formed from either or both organic and inorganic materials, including, but not limited to, microelectronic materials, whether electrically conducting, electrically semiconducting, or electrically insulating, including materials such as II-IV and IXI-V materials, oxides and nitrides, like S sN ⁇ AI2O3, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon®, or elastomers such as two- component addition-cure silicone rubber, and glasses,
  • a solid state support structure may be formed from monatomic layers, such as graphene, or layers that are only a few atoms thick such as those disclosed in U.S. Patent No. 8,898,481, and U.S. Patent Application Publication 2014/174927, both hereby incorporated by reference. More than one support layer material can be included, such as more than one graphene layer, as disclosed in US Patent Application Publication 2013/309778, incorporated herein by reference.
  • Suitable silicon nitride membranes are disclosed in U.S. Patent No. 8,627,067, and the support structure may be chemically functionalized, such as disclosed in U.S. patent application publication 2011/053284, both hereby incorporated by reference. [ ⁇ 064] '
  • any suitable method can be employed for producing a nanopore in the .support structure.
  • electron beam milling, ion beam milling, material sculpting with an energetic beam, dry etching, wet chemical or electrochemical etching, or other method can be employed for producing a nanopore, as described, e.g., in U.S. Patent Applicatio
  • a biological nanopore can be provided within a solid state aperture.
  • a biological nanopore may be a
  • the biological pore ma be a natarally occurring pore or may be a mutant pore.
  • Typical pores are described in U.S. Patent Application. No. 2012/1007802, and are described, in Stoddart D et aL Proc Natl Acad Sci, 12;lO6(19 ⁇ :7702-7, Stoddart D et aL, Angew Chem Int Ed Engl. 2010;49(3):556-9, Stoddart D et al., Nano Lett, 2010 Sep 8;10(9):3633 ⁇ 7, Butler TZ et al, Proc Natl Acad Sci 2008;lO5(52):2O847-52, U.S.
  • the pore may be homo-oligomeric, namely, derived from identical monomers.
  • the pore may be hetero-oligomeric, namely where at least one monomer differs from the others.
  • the pore may be a DNA origami, pore, as described by Langecker et ai, Science, 2012; 338; 932-936, hereby incorporated by reference.
  • the pore can be provided within an
  • An amphophilic layer is a layer formed from amphophilic molecules, such as phospholipids, which have bot hydrophiiic and lipophilic properties.
  • the amphophilic layer can be a monolayer or bilayer, with the layer selected from a lipid bilayer or non-natural lipid bilayer.
  • the bilayer can be synthetic, such as that disclosed by Kunitake T., Angew, Chem. Int. Ed. Engl. 31 (1992) 709-726.
  • the amphophilic layer can be a co-block polymer such as disclosed by Gonzalez-Perez et ah, Langmuir. 2009, 25, 10447-10450, and U.S. No 6,723,814, both hereby incorporated by inference.
  • the polymer can be, e.g., a PMOXA-PDMS-PMOXA triblock copolymer.
  • any of these support structures, nanopores, and electrical configurations fo measurin the local electrical potential at one or more sites in a nanopore sensor can be employed in a method for sensing the translocation of species through, the nanopore.
  • the nanopore sensor As a circuit 35 including electrical components corresponding to physical elements of the sensor, as show in Fig. 2A,
  • the cis and trans reservoirs can each be modeled with a characteristic fluidic access resistance, Rrmna, 36, Ra», 38, This access resistance is defined for this analysis as the fluidic resistance in a reservoir solution local to the site of the nanopore, not in the bulk solution away from the nanopore.
  • the nanopore can be modeled with a characteristic nanopore solution resistance, Rpore, 40 that is the fluidic resistance of solution through the length of the nanopore between the two sides of the support structure in which the nanopore is disposed.
  • the nanopore can also be modeled with a characteristic capacitance C , that is a functiono of the membrane or other support structure in which the nanopore is disposed.
  • the access resistance of both chambers and the nanopore solution resistance are variable.
  • the nanopore in a nanopore sensor starting condition in which no species are translocatin the nanopore, the nanopore can be characterized by the solution resistance, J3 ⁇ 4r1 ⁇ 2 «, given above, and both fluidic reservoirs ca be characterized by the access resistances of the trans reservoir and the cis reservoir, Rrmm and respectively.
  • a species object such as a biological molecule 45
  • the solution resistance, Rp m, of the nanopore and the access resistances, Rihm and of each of the reservoirs change because the molecule in the nanopore at least partially blocks the passageway through the nanopore length, changing the effective diameter of the nanopore.
  • the fluidic solution resistance of the nanopore and the access resistance of both reservoirs increase above the resistance of the nanopore and access resistance of both reservoirs with no molecule present in the nanopore.
  • the partial blockage of the nanopore by a species object effects the nanopore solution resistance and the reservoir access resistances differently, as explained in detail below.
  • the partial blockage of the naiiopore by a translocating species causes a corresponding redistribution of electrical voltage occurs between the nanopore and the eis and trans reservoirs solutions, and the electrical potential at sites throughout the
  • nanopore sensor accordingly adjusts.
  • the local electrical potential at both the sites denoted as A and B in Fig, 2A thereby changes accordingly with this change in nanopore solution resistance and redistribution of voltage between the reservoir solutions and the nanopore.
  • a measurement of electrical potentia l at either of these sites, or at another sit of the nanopore sensor configuration, or a measurement of a difference in local potential between two or more sites, thereby provides an indication of the translocatio of the molecule through the nanopore.
  • the local electrical potential at. a selected naiiopore sensor site and changes in this potential can be sensed by an electrical transduction element disposed in the nanopore sensor. For example, changes in the
  • the nanopore sensor arrangements of Pigs. 1A-1B correspond to a local electrical potential measurement at site A in the circuit 35 of Fig. 2 A
  • the nanopore sensor arrangements of Fig. IC-ID correspond to a local electrical potential measurement at site B in the circuit 35 of Fig. 2A.
  • the nanopore sensor arrangement of Fig, IE corresponds to a local electrical potential measurement at both sites A. and B in the circuit 35 of Fig. 2A, and enables a determination of the difference between the potential at. those two sites.
  • FIG. 2B An electrical circuit equivalent of the example confi uration of Fig. IB is shown in Fig. 2B.
  • the location of an electrical transduction element for measuring local potential e.g., the channel of a transistor 22, is here positioned at the site A in Fig. 2A. providing a local electrical potential indication in . the trans reservoir at the trans reservoir side of the nanopore.
  • measurement circuit can be monitored for changes in electrical potential, corresponding to changes in the state of the nanopore and the presence or absence of one or more objects in the nanopore.
  • an electrical transduction element such as a device, region of a device, circuit, or other transduction element that makes a local electrical potential measurement as species objects translocate the nanopore.
  • the nanopore sensor can be modeled as shown in the schematic representation of Fig. 3A.
  • Several assumptions can he employed to enable an analytical calculation.
  • the fluidic reservoirs are assumed to include electrically conductive ionic solutions.
  • the two reservoir solutions are specified to include distinct ionic concentrations that may be differing ionic concentrations.
  • the ionic concentration distribution through the nanopore system is determmed by the steady state diffusion that is driven by the cis / trans reservoir concentration difference: the diffusion is assumed to reach steady state.
  • a further assumption can be made by approximating the buffer concentration distributiori and electrical potential a being constant in small hemispheres o both sides of the nanopore.
  • the nanopore sensor is assumed to be in steady state. Under these conditions, the diffusion equations of the nanopore sensor are give as.:
  • I and d are thickness of the nanopore support structure and nanopore diameter, respectively. Because the ionic concentration distribution is therefore known and the solutio conductivity is proportional to the
  • the electrical potential equals the voltage applied across the structure or membrane, i.e., a transmembrane voltage (TMV), to electiOphoreticaiiy drive an object through the nanopore, and that far away from nanopore in the trans chamber, the potential is 0 V, then the voltages in the nanopore sensor, namely, the voltage in the cis reservoir, Vc(r), the voltage in the trans reservoir, 1 ⁇ 2 ⁇ r) . , and the voltage in the nanopore, V r) s are given as:
  • the electrical potential change at the trans reservoir side of the nanopore can be estimated by the electrical potential change due to a reduction in the nanopore area, A, by the presence of a species object, such as a molecule, i the nanopore, as:
  • the resist ances of the nanopore sensor namely, .&.3 ⁇ 4, JR * «, and Rpure, can he computed based on the above expressions for voltage drop across the reservoirs and the nanopore as:
  • the total resistance and ionic current of the nanopore sensor are gi ven as:
  • Figs. 3B-3E are plots of electrical potential and electric field in the nanopore, demonstrating these conditions.
  • the electrical potential in the nanopore as a functiono of distance from the nanopore opening at the cis reservoir is plotted in Fig. 3B, based on Expression (8) above. That same potential is plotted in Fig. 3C for a condition in which the cisitrans buffer solution concentration ratio is instead. 100:1. Note the increase in electrical potential at a given nanopore location for the unbalanced, buffer solution ratio at points closer to the lower-concentration reservoir.
  • Fig. 3D is a plot of the electric field, in the nanopore under the conditions given above, here for a balanced buffer solution ratio, based o
  • the ionic reservoir solutions can be provided with differin ion concentrations.
  • the local potential nieasurement is preferably made at a site in the reservoir which includes the lower ionic concentration.
  • the buffer concentration of the lower-ion concentration solution be selected to render the access resistance of that reservoir of the same order of magnitude as the nanopore resistance and much larger than the resistance of the high-ion concentration solution, e.g., at least an order of magnitude greater than that of the high-ion concentration solution, so that, for example, if the local potential measurement is being made in the trans reservoir:
  • Fig. 4 A is a plot of Expression (8) for a 50 nm- thick nanopore membrane and a configuration of a 1 V TMV for eleetrophoretic species translocation as a dsDNA molecule translocates through the nanopore.
  • the potential change is show as a function of the C JCfram ionic concentration ratio for various nanopore diameters below 10 nm.
  • Fig. 4B is a plot of the corresponding calculated potential change distributio in the trans reservoir for a 10 nm -diameter nanopore at 1 V TMV for the selected 100:1 CaJCrmm solution concentration ratio
  • the ratio of ionic fluid buffer concentration in the two reservoirs can be selected with the lower buffer concentration solution in the measurement reservoir, to maximize the amplitude of the electrical potential changes at that selected measurement site.
  • the distribution of this resulting potential change is highly localized within several tens of nanometers of the nanopore, as shown in Fig 4.B.
  • the local potential sensing technique produces a local potential measurement signal that depends on the trans-membrane voltage (TMV) and the ionic current signal
  • TMV trans-membrane voltage
  • Other sensor based nanopore technologies generally rely on a direct interaction between a translocating species and the nanopore sensor through, e.g., electrical coupling or quantum mechanical tunnelling.
  • the nanopore output signal is typically not directly related to the TMV or ionic curren and should not change significantly when the TMV is changed.
  • the nanopore sensor signal is proportional to the TMV and can be regarded as a linear amplification of the ionic current signal.
  • both, the local potential measurement signal and the ionic current signal amplitudes depend on the TMV linearly, but the ratio between them is a constant for a given nanopore geometry and reservoir solution concentrations, as evidenced by the expressions given above,
  • An advantage of the local potential measurement method is the characteristically high-bandwidth capability of the me suremen with low noise. Low signal bandwidth is one of the issues that limits direct nanopore sensing by the conventional ionic current blockage measurement technique, due to the difficulties of high bandwidth amplification of very small measured electrical current signals. This cars be particularly true for a small nanopore whe employed for DNA sensing.
  • a large local electrical potential signal is measured instead of a small current signal, so the signal bandwidth is not limited by the capabilities of a current amplifier.
  • high-bandwidth signal processing electronics can be integrated on a solid state nanopore sensing structure.
  • concentration ratio can be selected, in one embodiment, to optimize the signal bandwidth of the nanopore sensor. Given that the local potential
  • the cis reservoir solution concentration is set as high as reasonable, e.g., about 4 M, about a saturated solution, to minimize the nanopore solution resistance.
  • the signal noise as a function of bandwidth is nal zed, e.g., based on the plot of Fig. 40.
  • free space refers to a computation based on free-space molecular size.
  • Boyley refers to computation based on molecular size from previous work by Bayley et al, in J.
  • the nanopore is give as a 1 nm-diameter nanopore in a graphene membrane, with a 4 M cis reservoir solution concentration, a buffer
  • the dielectric loss factor for graphene is unknown, so 1 was used for convenience.
  • Finding the cross point of the signal and total noise in the plot sets the 1:1 signal-to-noise ratio (S/N). This is the highest possible signal bandwidth. For example, for the fiuidic nanopore operation, the 1:1 S/N ratio is at a bandwidth of about 100 MHz. A bandwidth greater than about 50 MHz can be preferred as well as the 100 MHz bandwidth.
  • the 100 MHz bandwidth corresponds to reservoir solution concentration ratio of about 50:1, where the local potential is to be made in the low-concentration reservoir side of the nanopore.
  • any reservoir concentration ratio higher than about 50:1 will decrease the nanopore sensor parameters used i this example.
  • the bandwidth can be optimised and there exists an optimisation point of reservoir concentratio ratio, say 50:1.
  • the reservoir solution concentration ratio therefore can be selected, in one embodiment, based on a trade-off between the characteristic noise of the nanopore sensor and the desired operational bandwidth of the iianopore sensor. It is to be recognized therefore that to minimize noise, the reservoir solution concentration ratio can be increased, but that the bandwidth may be correspondingly reduced.
  • electronic signal processing such as low-pass filtering, or other processing of the signal, can be employed.
  • a nanopore of smaller diameter produces a larger signal for a given species object to translocate through the nanopore.
  • the nanopore extent is preferably based on the molecule extent, and the tuning of the reservoir concentratio ratio is made accordingly,
  • the reservoir buffer solution concentration ratio can also be selected, in a further embodiment, to produce a signal decay length, measured from the site of the nanopore, that accommodates a selected local potential measurement device. It is recognized that, the deca length of the signal should be sufficiently large to accommodate the arrangement of a potential measurement device within the decay length.
  • Fig. 4E is a plot of signal decay length for a range of buffer concentration ratios, given that the local potential measurement is to be made on the trans reservoir side of the nanopore. The plot is based on the circuit model shown inset in the plot.
  • a nanopore sensor 100 provided herein, the nanopore sensor 100 including a cis reservoir 102 and a trans reservoir 104 on opposin sides of a nanopore 12 that is disposed in a support structure 14.
  • the nanopore must be traversed in a pat of fluidic communication between the two reservoirs.
  • a fluidic passage 105 is disposed between one reservoir, here shown as the trans reservoir, and the nanopore 12 to fluidieaiiy connect that reservoir to the nanopore through the fluidic passage.
  • the fluidic passage has a passage length that is greater tha passage cross-sectional extent, width, or diameter, and is connected to enable fluidic communication between the nanopore and the reservoir to which the passage leads.
  • the second reservoir here the cis reservoir, is arranged for fluidic communication with, the fluidic passage by way of the nanopore.
  • the second reservoir does not need to include a second fhiidie passage.
  • the nanopore sensor 100 of this embodiment can include electrodes 18, 15, and a voltage source 16 for applying an electrical potential between the reservoirs, across the solid state support, structu e.
  • the nanopore is disposed in the suitable support structure and is solid state, biological, or some combination of the two in the manner described above.
  • This nanopore sensor embodiment can be electrically modelled as shown in the circuit of Fig. 8. In this model, the aspect ratio of the nanopore, i,e. ⁇ the ratio of nanopore diameter and length, and the aspect ratio of the fluidic passage are a priori specified to be sufficiently large that the fluidic access resistance of the trans and cis reservoir chambers can be ignored.
  • an electrical transduction element 7. like that described above, is provided in the sensor to sense the electrical potential local to fluidic passage 105, to develop a characteristic that is indicative of the local electrical potential in the fluidic passage.
  • An electrical connection such as device or region of a device and/or circuit, a wire, or combination of circuit elements, that senses the electrical potential local to the site of the device and/or circuit can be provided as a transductio element to develop a signal indicative of local electrical potential.
  • the location of the electrical transduction element 7 can be in a reservoir, on a surface of the support structure, as shown in Fig. 5, at a location within the fluidic passage, or other location within the nanopore sensor.
  • a circuit 20 for supporting an electrical transduction element that is, e.g., a transistor device, having a source, S, a drain, D, and a channel region.
  • the channel region can be physicaiiy disposed at a location in the nanopore sensor environment to make a local electrical potential measurement. This physical location of the channel region of the transistor can be at any convenient and. suitable site for accessing local electrical potential.
  • V Se ns V 0 R FP /iR Pore + R FP ) (12) where Rpore is the resistance of the nanopore, RFP is the resistance of the fluidic passage, and Vo is the voltage 16 applied across the nanopore from the circuit .
  • translocation through the nanopore is the small voltage change that is caused by the correspondingly small resistance change of the nanopore due to the partial blockage of the nanopore by the translocating species, with Vsig given as:
  • Expression (1 ) is plotted in Fig. 7. This plot indicates that when the fluidic passage resistance is 10% of the nanopore resistance, the voltag signal, Vs «, is more than 30% of the maximum attainable signal.
  • a condition in which when the fluidic passage resistance, RFP, is at least about 1.0% of the nanopore resistance, RpaTM, and is no more than about 10 times the nanopore resistance is considered to be matching of the fluidic passage resistance and the nanopore resistance.
  • both the nanopore and the fluidic passage are generally cylindrical in geometry.
  • the access resistance of the cis and t ans reservoirs can be ignored for this analysis, given that the aspect ratio of the nanopore and the fluidic passage are relatively high relative to the cis and trans reservoirs.
  • diffusion of ionic concentrations between the cis and irons reservoirs is in a steady state condition if the ionic concentrations in the two reservoirs are different.
  • Figure 8 is a schematic view defining the geometry of the fluidic passage 105.
  • li, n and h, rs are the length and the radius of the nanopore and the fluidic passage, respectively.
  • Cc, Ci and CT are the ionic solution concentrations in the cis chamber, the ionic solution concentration at the interface between the nanopore and. the fluidic passage, and the ionic concentration in the trans chamber, respectively.
  • CFP concentration in the fluidic paseage
  • the ratio of ionic concentrations in the cis and trans chambers, CC/CT can be defined as Rc
  • the ratio of the fluidic passage radius to the nanopore radius, n/ri can be defined as R r
  • the aspect ratio of the fluidic passage is set as 300:1 for fluidic passage length to fluidic passage diameter, here 450 ⁇ deep, to match the nanopore resistance when a difference in ionic concentration of 100:1 is employed between cis and tram chambers.
  • Fig. 9 shows the resistance ratio for a 3:1 aspect-ratio nanopore, 100:1 ionic concentration difference, and assumptio of fluidic passage diameter of 1000 time nanopore diameter.
  • This nanopore arrangement including a fluidic passage connected between the nanopore and one of the reservOirs can compensate for some of the limitations imposed by only differing the cis and trans chamber ionic solution concentrations.
  • the ionic concentration affects the nanopore resistance as well as cis and trans reservoir access resistances, and in this scenario the ability to match the nanopore resistance with one reservoir access resistance is limited, accordingly limiting the voltage signal that can be produced.
  • fluidic access resistance is localized in the vicinity of the nanopore, and is a f unction of distance from the nanopore, so that for electrical transduction elements, such as an amphophilic membrane layer, that are mobile, there can be significant signal fluctuation.
  • the inclusion of a fluidic passage between the nanopore and one reservoir enables structural definition of a fluidic resistance and adds an additional control parameter to the nanopore sensor.
  • the nanopore sensor can be tuned to optimize voltage signal measurement. fOOlllJ
  • the fluidic passage-connecting- a reservoir to a nanopore can be configured in any convenient -arrangement that enables a selected aspect ratio and integration with the nanopore sensor.
  • the fluidic passage 105 can be formed as a well, a trench, a channel, or other fluidic holding chamber in a structure provided for defining the nanopore.
  • the iluiclic passage can be a duct, a channel, an open-ended trench, a path, or other geometry in a structure 120 that is arranged with a support structure 14 in which the nanopore 12 -is disposed.
  • a support structure layer 1 is disposed on a substrate 120 for providing the nanopore 12,
  • the fluidic passage is formed in the substrate 120.
  • the walls 115 of the fluidic passage can be configured to provide a suitable fluidic passage cross- sectional geometry, e.g., generally circular, elliptical, round, square, or other geometry.
  • the fluidic passage is connected to a reservoir, e.g., the trans reservoir, having any dimensionality; the figures represent the trans reservoir schematically to indicate that the trans reservoir has any dimensionalit and is not in general a high-aspect ratio passage like the fluidic passage.
  • the fluidic passage 105 can be disposed opposite a nanopore support structure.
  • a layer 125 of material can be provided on a nanopore support structure 14, with the fluidic passage 105 defined in the material layer 125.
  • a substrate or structure 120 that supports the nanopore support structure 14 can be provided opposite the fluidic passage, on the opposite side of the support, structure 14.
  • the fluidic passage configurations of Figs. 11-12 demonstrate that the fluidic passage can be disposed on any convenient location of the nanopore sensor.
  • the fluidic passage 105 can he provided as a population of pores, wells, channels, or other geometry, in a selected arrangement,
  • the fluidic passage 105 can include a layer of anodized aluminum oxide (AAO).
  • AAO is a conventional material that can be formed with ver high aspect ratio holes e.g., >10O0:1, having a diameter of about 100 rim. These AAO holes are arranged in a quasi-hexagonal lattice in two dimensions with a lattice constant in the range of several hundred nanometers.
  • An aluminum oxide film can be anodked under controllable anodizing conditions, and such anodization can be conducted on a film having surface pre- atterning, to tune the AAO lattice constant and hole diameter.
  • the population of AAO holes work in concert to provide an effective aspect ratio.
  • This AAO arrangement is an example of a holey film, membrane, or other structure having a population of holes, wells, pathways, or other channels, that can he employed as a fluidic passage.
  • any fluidic passage configuration can be. filled with a gel or other porous substance, or a selected material that is disposed in the fluidic passage to increase the fluidic resistivity of the fluidic passage.
  • the fluidic passage can be configured in a vertical arrangement, horizontal arrangement, or combination of horizontal and vertical geometries.
  • a fluidic passage including a vertical passage section 184 and a hor zontal passage section 138, Such arrangements can be provided in, e.g., a surface layer 132 on a support structure 14, or can be integrated into a substrate or other structural feature.
  • the fluidic passage 105 can be configured in a lateral or vertical geometry of any suitable path for achieving a selected fluidic passage aspect ratio. As shown in the schematic view in Fig.
  • ISA a path that winds around itself, either laterally or vertically, can be configured for achieving a selected fluidic passage aspect ratio.
  • a serpentine path either lateral or vertical, can be alternatively employed as a fluidic passage.
  • the geometry can be formed in a layer on a support structure, substrate, or combination of elements in the sensor.
  • the port 140 of the fluidic passage can be connected in the horizontal or vertical direction to provide fluidic
  • These various fluidic passage configurations can in various embodiments include, e.g., an AAO fil having holes of about 1.50 nm in diameter, with a hole-to-hole distance of about 300 am, and a film thickness of between about 100 pm - 1000 pm; a planar PDMS/oxide/nitride channel havin a width of between about 0.5 pm ⁇ I pm, a depth of between about 100 nm 200 nm, and a length of between about 200 pm 500 pm; a deep well in a dielectric material with a well diameter of between about 0.5 pm - 2 pm and a diameter of between about 20 pm - 50 pm; and a silicon wafer well having a diameter of between about 2 p.m - 6 pm and a depth of between about 200 pm — 300 pm.
  • the cis and trans ionic concentrations can be the same or can he different, with a selected ionic concentration ratio, e.g., between about 50:1 - 1000:1 as a ratio oici$:trans ionic concentrations.
  • a local electrical potential measurement can be made in the nanopore sensor wi h any suitable device or circuit that
  • the fluidic passage configurations described just above can be adapted to include an electrical transduction element at the site of the nanopore connection to the fluidic passage.
  • the configuration of Fig. 16A corresponds to the fluidic passage design of Fig, 11.
  • a silicon -on-insulator (SOI) wafer can be employed to form the substrate 120 with a buried oxide layer (BOX) and silicon layer 152 provided atop thereof.
  • the nanopore 12 can be formed in these layers 150, 152.
  • the silicon layer 152 can be configured as a conductance channel for making a local potential measurement at the site of the nanopore, with electrically conducting source 154 and drain 156 regions provided for measurin the conductance that is transduced by the silicon conductance channel. Similarly, as shown in Figs. 16B, a silicon layer 152 can be
  • a sensing electrode 158 or electrodes, of metal, carbon nanotubes, or other material can be configured at the site of the nanopore 12 and connected for sensing by, e.g.. a drain electrode 156.
  • lateral f!uidie passage channels are shown in cross section with a transduction element.
  • the fluidie passage 105 is provided in a layer 158, such as a nitride layer.
  • the nitride layer 158 is provided on a support structure 14 such as a silicon layer 152 from a SOI waver, with an underlying BOX layer 150.
  • Source and drain regions 154, 158 are provided in connection to the silicon layer 152, patterned as a channel for transducing the local electrical potential at the nanopore.
  • the fluidie passage shown in cross-section to depict a channel such as that in Figs.
  • I5A-15B is provided in a top layer 180, such as POMS.
  • the channel can be moulded into the top layer 160 and then connected to the nanopore sensor structure.
  • a silicon laye 152 from a SOI wafer can be configured as a conductance channel, with source and drain regions 154, 156, connected for transducin the local electrical potential at the nanopore.
  • nanowire -based FET device can be a well- suited device, but such is not required herein.
  • the SET, QPC, lipid bilayer, or other device and nanopore implemen ation, whether biological or solid state, can be employed. Any circuit or device that, enables a local potential
  • a nanowire FET can be configured at the site of the nanopore as shown in Fig, 17 here shown without the fl iridic passage for clarity.
  • the nanowire can be formed, of any suitable electrically conducting or
  • Nanowire refers to an electrical conduction channel that is characterized by a width that is compatible with the signal decay length measured from the nanopore site as described above. The channel width is preferably on the same order of magnitude as the decay length and can be larger.
  • the nanowire can be made from any semiconductor material that is stable in the selected reservoir solution.
  • Pig. 18 is a perspective view of an implementation 65 of the nanopore sensor of Fig. 6. Here is shown the nanowire 62 provided on a membrane support structure 14 that is self- supported across its extent, like a trampoline, between a support frame at. the edges, provided on a support structure 64 such as a substrate.
  • the nanowire is provided on the membrane with a nanopore extending through the thickness of the nanowire and the membrane. As shown in Figs. 17 and 18, the nanopore 12 does not extend across the width of the nanowire. There is a region of the nanowire that is unbroken along the extent of the nanopore so that electrical conduction is continuous along the length of the nanowire, A metallization region or other electrically conducting region is provided at each end of the nanowire to form source (S) and drain (D) regions.
  • the nanopore sensor can be configured with cis and trans reservoirs and a flttidic passage connected to one of the reservoirs, for detecting translocation of species from one reservoir through the nanopore to the other reservoir.
  • the support structure and nanowire configuration can be implemented in. variety of alterna ive arrangements, and a support structure, such as a membrane support layer, is not required for applications in which, a nanowire material is self supporting and can itself function as a support structure in which the nanopore is disposed.
  • a support structure such as a membrane support layer
  • a support layer 70 which in turn supports a graphene membrane 72.
  • the graphene membrane 72 is self- supported across a aperture in the support, laye 70.
  • the membrane in turn supports a nanowire 82, with a nanopore 12 extending through the thickness of the nanowire and the grapheme, and the nanowire remaining continuous along some point of the nanowire.
  • this arrangement can be altered, with the nanowire 72 instead disposed under the graphene layer 72, on a support layer 70.
  • a support structure such as a support layer 70, on which is disposed a graphene layer 68 that functions to provide a. structure in which a nanopore 12 is configured and that itself functions to provide a nanowire.
  • the graphene can be provided in any suitable geometr that, provides the requisite nanowire at the site of the nanopore 12, In this configuration, the graphene layer 88, due to its thickness and
  • any in a very wide range of electrical transduction elements that can be employed to measure the electrical potential local to the fluidic passage of the nanopore sensor.
  • a semiconductor-based PET or -other sensing device, a sensing metal electrode connected to a device such as an PET device, a graphene-based device, or other suitable transduction element can be employed.
  • the support layer, support layer membrane, nanowire, and support structure can be configured with any in a wide range of materials combinations and thicknesses.
  • the fluidic passage configurations described above can be integrated into any of these arrangements.
  • it can be preferred that the structure in which the nanopore is disposed be as thin as possible, and preferably no thicker than the extent of a. species object, or object region to be detected.
  • support structure materials can include nitrides, oxides, conductors, semiconductors, graphene, plastics, or other suitable material which can be electrically insulating or electrically
  • the nanopore is provided at the location of a nanowire 62 such that an unbroken, continuous path for electrical conduction is provided through the nanowire.
  • the nanopore can be provided at a central region of the nanowire, as depicted in Fig, 22A, can be provided at an edge of the nanowire, as depicted in Figs. 22B-22CX or can be provided at a site near to or adjacent to the nanowire, as depicted in Fig. 22D. In all cases, a continuous path for electrical conduction is provided through the nanowire.
  • a continuous path for electrical conduction is provided through the nanowire.
  • the sensitivity of the nanopore region is also significantly enhanced compared to the sensitivity of the same region prior to nanopore drilling.
  • This sensitivity localization can be understood by a model accounting for the reduction of the cross-sectional area of the nanowire as a conduction channel, assuming all other material properties, such as doping level and mobility remain unchanged.
  • the reduced cross-sectional area of the nanowire increases the resistance of the nanopore region and therefore alleviates series resistance and signal attenuation from othe portions of the nanowire.
  • this sensitivity enhancement at the nanopore region can be obtained from the following equation for a rectangular-shaped nanopore as an example:
  • A is the sensitivity enhancement defined as the sensitivity of the device with a nanopore divided by the sensitivity without the nanopore, and po and p are the linear resistivities of the nanowire conduction channel with and without the nanopore, respectively, L i the total channel length and ho is the channel length of the nanopore region, which for thi square example is equal to the side length of the nanopore along the nanowire axial direction.
  • L i the total channel length
  • ho the channel length of the nanopore region, which for thi square example is equal to the side length of the nanopore along the nanowire axial direction.
  • a short-channel nanowire can be preferred, and for many applications, a silicon nanowire (SiNW) can be preferred because the SiNW lias been demonstrated as an excellent electrical potential and charge sensor for sub -cellular and single- vims level signalling with remarkable stability i solution.
  • SiNW channel can be reduced, if desired, to less than about 200 nra by nickel solid-state diffusion.
  • SiNWs can be fabricated by, e.g., chemical vapor deposition, or other suitable process, and disposed on a selected membrane, such as a nitride membrane, by solution.
  • a selected membrane such as a nitride membrane
  • Electron beam lithography or optical lithography can be employed for producing source and drain electrodes at ends of the nanowire. All electrodes and electrical contacts are to be passivated with, e.g., a nitride or oxide material, and such can be accomplished after metal evaporation and before lift-off processes.
  • the nanopore can be produced at a selected site by, e.g., electron beam, or by other beam specie or etching process that produces a selected nanopore dimension.
  • a membrane such as a nitride membrane
  • a micron-sized aperture in the membrane e.g.. by electron beam lithography or photolithography and reactive ion etching
  • a graphene sheet or piece is disposed on the nitride membrane, covering the aperture, to form a graphene membrane.
  • the graphene sheet can be synthesized b CVD or other process, or produced by mechanical exfoliation, and transferred to the nitride membrane, over the nitride membrane aperture, 1321 Electron beam lithography or photolithography can then be conducted with metal evaporation to define electrodes in the conventional manner on the nitride membrane. Dielectrophoresis or other suitable process can then be employed to align a nanowire, such as a silicon nanowire, on top of the graphene membrane at the location of the aperture in the nitride
  • Electron beam lithography or photolithography can then be conducted with metal evaporation to define the source and drain contacts at ends of the SiNW. Thereafter, excessive graphene can be removed by electron beam lithography or photolithography and, e.g., UV-ozone stripper, oxygen plasma, or other suitable method to remove graphene from regions outside the intended graphene membrane location. Finally, a nanopore is produced through a site at the nanowire and the underlying graphene membrane by, e.g.. electron beam milling, ion beam milling, etching, or other suitable process as described above.
  • a suitable structure can he employed for configuring the arrangement, e.g.. with a silicon-on- insulator chip (SOI).
  • SOI silicon-on- insulator chip
  • an aperture is first formed through the backside thick silicon portion of the SOI chip, e.g., by Xl3 ⁇ 4 etching, stopping on the oxide layer, to form an oxide-silicon membrane.
  • electron beam lithography or photolithography is employed to remove the oxide layer from the SOI chip in a smaller aperture region, producing a membrane of silicon from the thin silicon regio of the SOI chip.
  • This silicon mem brane is then etched to form a nanowire of silicon, e.g.. with electron beam lithography o photolithography and chemical etching or RIE.
  • a dove-tail-shaped Si piece is formed as shown in Fig. 20B, aligned with the aperture in the oxide membrane of the SOI chip.
  • Electron beam lithography or photolithography can then be conducted with metal evaporation to define electrodes in the conventional manner on the oxide layer. Then a graphene sheet or piece is disposed on the oxide membrane, covering the aperture, to form a graphene membrane over the silicon nanowire.
  • the graphene sheet can be synthesized by GVD or other process, or produced by mechanical exfoliation, and transferred to the oxide membrane, over the SiNW and oxide membrane aperture. It is recognized that because the graphene sheet is bein overlaid on top of the patterned silicon layer, the graphene piece may not he fiat. If leakage is a concern for this configuration, then a thin layer of, e.g., SiO s can be coated around the graphene edges to form a. sealed edge condition.
  • a membrane such as a nitride membrane
  • a graphene sheet or piece is disposed on the nitride membrane, covering the aperture, to form, a graphene membrane.
  • the graphene sheet can be
  • Electron beam lithography or photolithography can then be conducted with metal evaporation to define source and drain electrodes in the conventional manner on the graphene membrane. Thereafter, the graphene is patterned in a dovetail or other selected shape by electron beam lithography or photolithography and, e.g., tJV-ozone stripper, oxygen plasma, or other suitable method to produce a narrow graphene region in the vicinity of the selected site for a nanopore. Finally, a nanopore is produced through the graphene membrane by, e.g., electron beam.
  • any suitable membrane material can be employed.
  • a nitride membrane structure or other structure can be employed, such as a graphene membrane or combination graphene- nitride membrane structure as-described above.
  • an electrically conducting membrane material it can be preferred to coat the material with an insulating layer, such as an oxide or nitride layer, on the side of the membrane on which the SET is to he formed. Electro beam lithography and metal evaporation techniques can then be employed to form the source and drain regions and the SET region out of a suitable metal.
  • a nanopore can then be formed at the location of the SET in the manner given above. If an insulating layer is provided on an electrically conducting membrane material and the insulating layer coated the length of the nanopore through the
  • insulating material from the nanopore sidewall by, e.g., HF or other suitable etching, from the backside of the nanopore, to remove the insulator layer from the nanopore and from the adjacent vicinity of the nanopore.
  • an SOI structure can be employed, removing the thick silicon layer in the manner described above, and then using electron beam lithography to define the top silicon layer structure in the QPC arrangement.
  • the nanopore can then be formed through the membrane in the manner given above.
  • a planar channel can be defined by patterned etching of a nitride or oxide layer at the nanopore site, with an oxide, glass, PDMS, or other material bonded onto the nitride or oxide laye to seal the fluidic passage channel.
  • a thick material layer such as an oxide layer, nitride layer, PDMS layer, polymer or other layer, can be etched or drilled, e.g., by deep reactive ion etching (RIE) to define a fluidic passage.
  • RIE deep reactive ion etching
  • an underlying support substrate such as a silicon wafer
  • HIE HIE
  • a fluidic passage through the thickness of the wafer can be etched, e.g., by HIE, to form a fluidic passage through the thickness of the wafer.
  • Any suitable support structure material and device material can be employed.
  • the nanopore sensor fabrication processes can be tailored to accommodate any suitable nanopore structure, whether solid state, biological, o some combination of the two.
  • a protein nanopore disposed in an aperture of a solid state support structure such as an PET channel material as described above.
  • a protein nanopore can be employed as disposed in an araphiphilic layer, or aperture in a solid state support structure at the site of an FET channel. Any combination of materials can be employed in the support structure that contains the nanopore,
  • th dimensions of the nanopore be selected based on a selected ratio of the reservoir buffer solution concentrations, to achieve a desired electrical potential measurement in the manner described above, in conjunction with consideration for the species objects to be investigated with the nanopore sensor.
  • the analytical expressions above can be employed to determine an optimum nanopore size for a give species to be detected by translocatio through the nanopore, in concert with the other nanopore sensor parameters and operation, for enabling electrical potential measurement, for nanopore sensing of the species.
  • the graphene-based nanopore sensors described above are particularly attractive for sensing molecular species such as DNA and other biopolymer species because the graphene thickness is on the order of a DNA base extent. But because graphene is electricall gated on both sides of the graphene by the ci$ and trans reservoir solutions, and the electrical potential i the two reservoirs is opposite, the sum of electrical potentials that is indicated by the graphene potential measurement is smaller than that indicated by the implementation of a nanowire on one side of a membrane. But for a small nanopore, e.g., of about 1 am in diameter, and with a
  • the sum of electrical potentials that is indicated by the graphene potential measurement is comparable to that of a nanowire nanopore sensor.
  • the rate of translocation can be controlled by a polymer binding moiety, for a graphene-based nanopore sensor or other nanopore support structure material.
  • the moiety can move the polymer through the nanopore with or against an. applied field.
  • the moiety can he a molecular motor using for example, in the ease where the moiety is an enzyme, enzymatic activity, or as a molecular brake.
  • the polymer is a polynucleotide there are a number of methods for controlling the rate of translocation including use of polynucleotide binding enzymes.
  • Suitable enzymes for controlling the rate of translocation of polynucleotides include, but are not limited to, polymerases, l elicaseSv exoxiueleases. single stranded and double stranded binding proteins, and topoisomerases, such as gyrases.
  • the enzyme may be a helicase or modified helicase such as disclosed by WO2013-057495 and
  • WO2014-01326Q For other polymer types, moieties that interact with that polymer type can be used.
  • the polymer interacting moiety may be any disclosed in. WO -2010/086603, WO-2012/107778, and Lieherman KR et al J Am Chem See, 2010:132(50); 17961-72), and for voltage gated schemes.
  • the signal may be referred to as being k-mer dependent, a fe-mer being k polymer units of a polymer, where k is a positive integer.
  • the extent to which the signal is dependent upon a k-mev is dependent upon the shape and length of the aperture and the polymer type. For example, with the translocation of a polynucleotide through a MspA pore, the signal may he considered as being dependent upon 5 nucleotide bases.
  • the signal may he dependent upon only a small number of polymer units and may even be dominated by a single polymer unit.
  • the measured signals may be used to determine a sequence probability of polymer units or to determine the presence or absence of an analyte. Suitable exemplary methods of signal analysis are disclosed in WO2013-041878 and W02O13- 121224.
  • SiNW FET Fabrication of a SiNW FET in a Nanopore Sensor 001461 SiNWs were synthesized using an Au-nanoparticle-catalyzed chemical vapor deposition (CVD) method. 30 iim-diameter gold nanopartieles (Ted Pella Inc., Redding, CA) were dispersed on a silicon wafer coated with a 800 nm-thick layer of silicon oxide (NOVA Electronic Materials Inc., Flower Mound, TX). Boron-doped p-type SiNWs were synthesized at 435°C and 30 torr, with 2,4 standard cubic centimeters per minute (seem) siiane as a silicon source. 3 seem diborane (100 ppm in.
  • CVD chemical vapor deposition
  • the structure was then loaded into a field emission transmission electron microscope (TEM) (JEOL 2010, 200kV) and a nanopore of about 9 nm or 10 am in extent was drilled by through the nanowire at a selected location by convergent high energy electron beam into one spot for approximately 2 - 5 minutes.
  • TEM field emission transmission electron microscope
  • the nanopore was sited at the edge of the nanowire, as depicted in the arrangement of Pig. 22B, whereby a substantial portion of the nanowire width was continuous.
  • Si W FET sensor of the nanopore sensor was characterized by scanning gate microscopy (SGM).
  • SGM scanning gate microscopy
  • a SiNW FET device was fabricated in accordance with the method of Example I, here with ⁇ 2 ,um long channel length to accommodate the limited spatial resolution of SGM.
  • SGM was performed in a Nanoscope Ilia Multi-Mode AFM (Digital
  • nanowire as a function of the position of a - 10 V biased conductive AFM tip (PPP-NCHPt, Nanosensors Inc., Neuchatel, SW).
  • the AFM tip was 20 nm above the surface during SGM recording.
  • Fig. 23 is a plot of sensitivity, defined as conductance change divided by AFM tip gate voltage, along the nanowire before nanopore formation and after nanopore formation. It is clear that the sensitivity of the device is sharpl localized and aligned with the nanopore. More importantly, the sensitivity of the nanopore region is also significantly enhanced compared to the sensitivity of the same region prior to nanopore formation.
  • Example III
  • PDMS chambers were sonicated first in DJ. water, then 70% ethanol and finally pure ethanol, each fo ⁇ 30 minutes and then stored in pure ethanol. Just before assembly, PDMS chambers were baked in a clean glass petri dish at ⁇ 80 °C for ⁇ 2 hours to remove most of the absorbed ethanol.
  • PCB chip carrier was produced for makin electrical connectio to the nanopore sensor, and was cleaned by Seotch-Brifce ⁇ 3M, St. Paul, MN) to remove the copper surface oxide and any contaminants such as glue.
  • the PCB was then sonicated in isopropyl alcohol and then in 70 % ethanol, each for ⁇ 30 minutes.
  • Gold solution electrodes were cleaned in piranha solution for ⁇ 1 hour just before assembly.
  • the cleaned nanowire-nanopore structure was glued into a ⁇ 250 ⁇ -deep center pit of the PCB chip carrier using wik-Cast (World Precision Instruments, Inc., Sarasota, FL) silicone glue, with the device side surface approximately flush to the surface of the rest of PCB chip carrier.
  • the source and drain electrical contacts of the device ' were wired to copper fingers on the chip carrier by wire bonding (West-Bond Inc., Anaheim, CA).
  • the front PDMS chamber was formed of a piece of PDMS with a ⁇ 1 ,8 mm hole in the center, with a protrusion of ⁇ 0.5 ram around one side of the hole opening, for pressing against the nanopore membrane surface to ensure a tight seal.
  • the PDMS chambers were mechanicall -clamped onto both sides of the chip carrier and Au electrodes were inserted through the PDMS reservoirs.
  • the gold electrodes function as electrical connections for biasing the PDMS chamber solutions to produce a transmembrane voltage (TMV) for driving species translocation through the nanopore electrophoretically.
  • TMV transmembrane voltage
  • 015 1 The trans chamber was selected as the reservoir in which potential measurements would be made for the nanopore sensor. Thus, the assembly was arranged with the membrane oriented such that, the nanowire was located facing the trans reservoir.
  • the trans chamber was filled with a solution having -a concentration of ⁇ 10 mM buffer, with 10 mM KC1 + 0.1 -x TAE buffer: 4 mM tris-acetate and 0,1 mM EDTA solution.
  • the cis chamber was accordingly filled with a higher ionic concentration solution to provide the requisite reservoir concentration ratio to provide a higher assess resistance at the site of local potential measurement, in the trans chamber.
  • nanowire-iiauopore structure produced by th method of the examples above and assembled with the solutions having buffer concentrations as prescribed by Example III was operated for sensing translocation of species objects, namely, double stranded DNA molecules of 1.4 nM pUC19 (dsDNA), Both the ionic current through the nanopore and the current from the nanowire FET device were measured.
  • species objects namely, double stranded DNA molecules of 1.4 nM pUC19 (dsDNA)
  • the nanowire FET current was amplified by a DL 1211 current amplifier (DL Instruments) with a 10 e
  • nanopore ionic current and nanowire FET signals were fed into a 144 OA digitizer, and recorded at 5 kHz by a computer. Operation of the nanopore sensor was carried out in a dark Faraday cage. To avoid 80 Hz noise that could be introduced by the electrical grounding from different instruments, the ground line was removed from all current amplifiers and all instruments (Amplifiers and digitizer) and the Faraday cage and were manually grounded to the building ground together.
  • Fig. 24A includes a plot, ⁇ ⁇ of the measured ionic current through the nanopore, and a plot, i of the measured nanowire FET conductance for a 2.-0 V TMV.
  • Fig. MB shows a plot, i, of the measured ionic current through the nanopore, and a plot, ii, of the measured nanowire FET conductance for a 2.4 V TMV.
  • the local potential measurement sensing method perfectly tracks the sensing by conventional ionic current measurement.
  • the local potential measurement method thereby enables the determination of the time of and the duration of translocation of an object through the nanopore.
  • a nanowire-nanopore structure was configured following the procedure in Example 111 above, but here with both cis and trans chambers filled with 1 M KC1 buffer instead of solutions having differing buffer concentrations. Operation of the nanopore sensor was then conducted with dsBNA provided in the trans reservoir- following the procedure of Example IV above, with a TMV of 0.6 V. The ionic current through the nanopore was measured, as was the local potential, via nanowire FET conductance , in the manner of Example IV. [00160] Fig.
  • 24C provides plots including plot i f of the measured ionie conductance and ii, of the measured FET conductance. As shown in the plots, translocation events were sensed by changes in ionic current when the TMV reached 0.5-0.6 V but the simultaneously-recorded FET conductance change was negligible at that voltage. The reservoir solution concentration ratio is therefore understood to play an important role in the signal generation.
  • the electric field through the nanopore is constant, as shown in the plot of Fig. 3D.
  • the reservoir concentrations are different, e.g., the 100:1 concentration of the examples above, then the electric field through the nanopore is smaller on the cis reservoir-side of the nanopore.
  • nanowire nanopore sensors were constructed following the methods of the examples above.
  • the three nanopore sensors were integrated with a common reservoir system, with a 1 M KC1 buffer solution in the eis chamber and a 10 niM KC1 buffer in the trans chamber, A transmembrane voltage of 3 V was employed, and 1.4 nM of pUCl9 DNA was provided for translocatio through the nanopores.
  • Fig. 25 provides plots i - iv of total ionic current and the nanowire FET conductance of each of the three nanopore sensors, respectively, during DNA nanopore translocation ' operation. As shown in the plots, continuous translocation events are observed in all three nanopore sensors as well as the total ionic current channel. All nanopore sensors operated independently and every falling or rising edge apparent in the ionic current channel can be uniquely correlated to a corresponding edge in one of the three nanopore sensors. Using the falling and rising edge of signals from all three nanopore sensors to reconstruct the total ionic current trace, the reconstruction is nearly perfect for of ail events. This nanopore operation demonstrates that a key advantage of the nanopore sensor is the large scale integration capability. Multiple independent nanopore sensors can be implemented without need for complex miero-fluidic systems.
  • a nanowire nanopore sensor was constructed following the methods of the examples above. Included was a 1 pm-diameter, 50 pm-long fluidic passage in a dielectric material connected between a fluidic reservoir and the nanopore. In the nanowire was disposed a protein nanopore in a lipid bilayer in an aperture of about 100 nm in the nanowire channel. The protein nanopore had an effective geometry of 1.5 nm in diameter and 4.5 nm in length. An ionic solution of 1.6M was provided in the cis reservoir and an ionic solution of ImM was provided in the trans reservoir. A 300 mV bias was applied across the nanopore for electrophoretieaily driving ssDNA through the nanopore.
  • the electrical potential of the fluidic passage, at the bottom of the passage was measured to he 150 mV and the voltage across the nanopore was 150 mV.
  • the effective nanopore diameter was 1.12 nm
  • the electrical potential at the bottom of the fluidic passage was measured to foe 128 mV, with the voltage across the nanopore being 172 mV.
  • the measured voltage signal corresponding to ssDNA translocation was 22 mV. This signal was distributed uniformly at the bottom of the fluidic passage, at the PET sensing surface, and did not change as the biological nanopore moved within the lipid membrane,
  • the nanopore sensor can provide sensing of species translocating through a nanopore and can discriminate between differing objects, such as DNA bases, as those objects translocate through the nanopore.
  • the nanopore sensor is not limited to sensing of a particular species or class of species and ca be employed for a wide range of applications. It is recognized that the nanopore sensor is particularly well-suited for sensing of biopolymer molecules that are provided for translocation through the nanopore. Such molecules include, e.g., nucleic acid chains such as DNA strands, an
  • oligonucleotide or section of single-stranded DNA nucleotides, nucleosides, polypeptide or protein, amino acids in general, or other biological polyme chain.
  • species object to be sensed by the nanopore sensor there is no particular limitation to the species object to be sensed by the nanopore sensor. With differing reservoir solution concentrations, a fluidic passage configuration, or some combination of these two features, it is

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Abstract

L'invention concerne un capteur à nanopore comportant un nanopore disposé dans une structure de support. Un passage fluidique est disposé entre un premier réservoir fluidique et le nanopore pour établir une communication fluidique entre le premier réservoir fluidique et le nanopore au moyen du passage fluidique. Le passage fluidique a une longueur de passage qui est supérieure à la largeur de passage, un second réservoir fluidique est en communication fluidique avec le nanopore, le nanopore assurant une communication fluidique entre le passage fluidique et le second réservoir. Des électrodes sont connectées pour imposer une différence de potentiel électrique à travers le nanopore. Au moins un élément de transduction électrique est disposé dans le capteur à nanopore avec une connexion pour mesurer le potentiel électrique qui est local pour le passage fluidique.
PCT/US2016/016664 2015-02-05 2016-02-04 Capteur à nanopore comprenant un passage fluidique WO2016127007A2 (fr)

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